Accurate quantification of DNA is a starting point for guaranteeing quality in molecular biological applications most of which mainly analyze DNA. Quantification of trace amounts of DNA is of special importance in certain analytical applications in which the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. Within this category are forensic DNA analysis, the detection and quantification of pathogenic agents, and the quantification of residual DNA impurities in biopharmaceutical products. Due to technical difficulties concerning quantification of trace-level DNA, special guidelines are often suggested to minimize analytical uncertainties and achieve a standard of best practice for the quantification of trace-level DNA. For example, the Food and Drug Administration (FDA) guidelines suggest that the acceptable residual amount of host cell DNA in biopharmaceutical drugs should be below 100 pg/dose, while the acceptable limit of host cell DNA allowed by the World Health Organization (WHO) and the European Union (EU) is up to 10 ng/dose.
Many different methods for quantifying DNA have been developed and applied for specific uses. UV spectrophotometry reading absorbance at 260 nm is the most common laboratory approach for quantifying DNA. However, it is hard to achieve the very high sensitivity required when quantifying practical samples containing only trace amounts of DNA. In addition, contamination by nucleotides, RNA, and proteins significantly interferes with the UV absorbance-based quantification of DNA. Fluorescence-based techniques are also widely used to quantify DNA. Given proper calibration standards, these methods show much higher sensitivity and accuracy compared with using UV spectrophotometry to quantify DNA. However, the fluorescence-based methods are also subject to interference by contaminants, and have been reported to be ineffective for quantifying amounts of DNA samples of less than 4 pg.
Several other methods were developed for a specific purpose regarding the quantification of an extremely low level DNA, especially for the quantification of residual host cell DNA in biopharmaceuticals. The hybridization method relies on radio isotopic or chemiluminescent detection of DNA hybridized to random and sequence-specific probes. Another method known as the ‘threshold method’ utilizes antibody-mediated detection and quantification of DNA captured by single-strand binding protein (SSB). Both the hybridization method and the threshold method are sufficiently sensitive to quantify picogram levels of DNA. These methods are advantageous in that they can quantify DNA in a sequence-independent manner and are applicable to universal DNA species. However, they also have disadvantageous including a relatively long analysis time, labor-intensiveness, and complicated procedures.
Another common platform for analyzing a trace amount of DNA is PCR, especially real-time PCR. Thanks to its extreme sensitivity and simplicity of experimentation, PCR technology has become the first choice for both qualitative and quantitative analysis of DNA in the lab. Although sequence-specificity is an incomparable merit of PCR technology, it also involves several important limitations with regard to the quantitative analysis of DNA. PCR will amplify and quantify only a specific target DNA, and not the whole DNA content. The quantity of the entirety of the DNA content therefore cannot be measured directly by PCR, but can only be estimated indirectly from the quantity of a specific target DNA. The sequence-specificity of PCR also limits the applicability of the method only to DNA samples containing more than one genome-equivalent amounts. The amount of human genomic DNA which allows individual genes to exist as at least one copy corresponds to 3 pg. Thus, even if the most effective reaction is performed under conditions ideal for PCR, the quantification limit by ordinary PCR will be 3 pg or above for human genomic DNA. When its performance is taken into consideration, ordinary PCR has a quantification limit of tens of picograms of mammalian genomic DNA. PCR is sensitive enough to allow the quantification of femtogram amounts of DNA from viruses and bacteria, but not sensitive enough to sufficiently quantify the DNA from mammalian cells. New approaches to amplifying multi-copy genes such as rDNA genes and Alu repeats have been applied to overcome the limited sensitivity of ordinary PCR with respect to mammalian cells. However, the approaches still have limitations because they are based on the assumption that the whole genome amount is distributed in a non-biased manner and a target multi-copy gene has a consistent copy number over the whole genome, which is impossible to achieve under ordinary analysis conditions. Conventional quantification technologies for trace amounts of DNA are summarized in Table 1, below.
TABLE 1HybridizationThresholdPCRSpecificityRandomSing strandedTargetsequenceDNA,sequenceSpeciesNon-speciesspecificspecificspecificityMinimal50600150detectionlength(bp)Resistance to++++interferenceTime48 6 2Sensitivity6 pg3 pg<1 pg(source [T. Wolter, A. Richter, Assays for controlling host cell impurities in biopharmaceuticals, Bioprocess Int. 3 (2005) 40-46])
Therefore, it is very important to develop a sensitive and universal method for quantification of femtogram levels of DNA.